Identifying spontaneous DNA lesions and how cells cope with them remains a significant challenge for biomedical research. The proposed project re-directs the applicant's past interest in prokaryotic extremophiles to address basic mechanisms of genome stability in the model eukaryote Saccharomyces cerevisiae. The project examines an unusual class of spontaneous mutations that result when DNA replication in S. cerevisiae is blocked by an unidentified lesion at particular guanine residues. In the absence of nucleotide excision repair, the trans-lesion DNA polymerase Pol zeta replicates past the unrepaired lesion, generating two mutations: one at the damaged guanine, and another 1-4 bases beyond. The available data further suggest that the unidentified lesion triggering these complex mutations could be a DNA-protein cross-link (DPC) generated by reactive oxygen species (ROS) during aerobic growth, and the proposed research will test predictions of this hypothesis. Fluctuation tests will determine whether the complex, Pol zeta-dependent mutations increase in response to chemical treatments that generate ROS or DPCs, or to mutations that inactivate specific ROS-defense mechanisms of S. cerevisiae. Other experiments will evaluate the ability of certain chemically defined DNA adducts, synthesized in vitro, to reproduce the mutations in vivo, and will develop improvements in the genetic assay for the mutations. The results have relevance for human health, because corresponding DNA lesions are expected to occur in human cells but have not been specifically identified, presumably due to lack of sufficiently sensitive detection methods. Genetic analyses in S. cerevisiae are versatile and precise, and provide the sensitivity needed to reveal the molecular nature of these DNA lesions in living cells. Pinpointing the DNA damage that triggers these spontaneous mutations in S. cerevisiae will enable future work to determine environmental and genetic risks to humans and the relevance to cancer and other diseases associated with genetic instability.